| Zwittermicin A(ZwA) is a novel,broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis.It is highly active against a wide range of plant pathogen as well as facilitates the insecticidal activity of the protein toxin produced by Bacillus thuringiensis.However,only part of its biosynthesis cluster has been identified and the biosynthesis pathway remains unclear.A bacterial artificial chromosome(BAC) library of Bacillus thuringiensis subsp,kurstaki strain YBT-1520,a ZwA-producing strain,was constructed.This library consists of 1536 clones,with an average insert size ranging from 24.5 kb to 72.5 kb.Assuming the size of strain YBT-1520 genome is 6 Mb,the library would have a 22.37-fold coverage of the YBT-1520 genome and the probability of the library covering any particular 1 kb gene is 99.9%.A 102kb CONTIG,which overlap the known Zwittermicin A biosynthesis related 16 kb DNA fragment,was constructed by PCR screen.ZwA biosynthesis cluster was identified based on the BAC clones terminal sequence result and the strain YBT-1520 genome sequence result.Southern blot result showed that ZwA biosynthesis cluster located on the chromosome of strain YBT-1520.This 67 kb cluster contains 30 open reading frames(ORFs).Except for the 8 genes locate in the announced 16 kb DNA fragment,there are one another polyketidal synthetase(PKS) coding gene orf1,3 nonribsomal peptide synthetase(NRPS) coding genes orf3,orf4,orf5,and the 2,3-diaminopropionate biosynthesis genes zwa5A and zwa5B,the modification enzyme carbamoyltransferase coding gene carbamoyltransferase, the post-transcript control factor coding gene phosphopantetheinyl transferase zwa9,and the transport related genes zwa7A and zwa7B.ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase respectively.The zwa5A~- mutant could not produce ZwA when zwa5A, the 2,3-diaminopropionate biosynthesis related gene,was interrupted by homologous exchange,while the zwa5A~- mutant resumed to produce ZwA when 2, 3-diaminopropionate was fed.It indicates that zwa5A gene is necessary for the biosynthesis of ZwA and 2,3-diaminopropionate can replace zwa5A in the biosynthesis of ZwA.The adenylation domain ZWAA in the ZwA biosynthesis cluster was over expressed in E.coli by pGEX-6P-1.ATP-dependent PPi exchange experiment result showed that ZWAA2 can active 2,3-diaminopropionate.Taken together,we have demonstrated that 2,3-diaminopropionate is one of the precursors during the biosynthesis of ZwA.Based on the announced hypothesis and our experiment results,we deduced that the premature ZwA is biosynthesized by a NRPS-PKS-NRPS(ORF2-ORF1-ORF3) hybrid pathway from five kinds of precursors:L-serine,malonyl-CoA,aminomalonyl-ACP, hydroxymalonyl-ACP and 2,3-diaminopropionate.Then carbamoyltransferase(ZWA6) catalyzes the premature ZwA into ZwA.The assembly of nonribsomal peptide(NRP )initiation factor L-serine and the polyketide(PK) elongation factor malonyl-CoA is catalyzed by ORF2,the assembly of PK elongation factors aminomalonyl-ACP and hydroxymalonyl-ACP is catalyzed by ORF1,while the assembly of NRP elongation factor 2,3-diaminopropionate is catalyzed of ORF3. L-serine-malonyl-CoA is the hybridization of NRPS-PKS(typeâ… ,catalyzed by one synthetase) while hydroxymalonyl-ACP-2,3-diaminopropionate is the hybridization of PKS-NRPS(typeâ…¡,catalyzed by two synthetases).The expression of the N-acyl-homoserine-lactonase coding gene Aii in ZwA-producing B.cereus UW85 and UW56,and its influence on the yield of ZwA were investigated in this study too.Western Blot and HPLC analysis results showed that Aii gene expressed in UW85 and UW56 successfully and did not impact on the ZwA yield.It demonstrates that B.cereus UW56,a ZwA high-producing strain,is a potential candidate in developing gene engineered strain to prevent Erwinia virulence.
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